PROJECT SUMMARY Lysosomal storage diseases (LSDs) are a large group of disorders that are caused by the absence of functional enzymes used for the degradation of substances within lysosomes. Fabry disease is the second most prevalent LSD in the United States and worldwide. It is characterized by the absence of functional ?-galactosidase A (GLA) enzymes that breaks down globotriaosylceramide (GL3) substrate, causing the latter to accumulate. In addition to causing debilitating pain, GL3 accumulation is particularly damaging to the kidneys and heart requiring the need for cardiac procedures, dialysis, and kidney transplants. Currently, treatment for Fabry disease in the United States consists of GLA enzyme replacement infusions. Although these biweekly infusions are clinically effective in arresting renal and cardiac organ damage, intravenous treatments are inconvenient, resource-intensive, time-consuming, and expensive. Furthermore, current treatments bear pharmacological drawbacks such as short enzyme plasma half-lives (45 min to 2.5 h) and infusion-related reactions (fever, chills, vomiting, hypotension, paresthesia) associated wit the administration of large single doses. Unfortunately, the development of enzyme-replacement therapies (ERTs) for Fabry has remained stagnant for the past decade, and current efforts are focused on biosimilars that lack additional clinical or quality of life benefits. The aim of this research proposal is to optimize the formulation of a prototype subcutaneous ERT that can be easily self-administered in multiple discrete doses. The prototype formulation to be optimized consists of GLA enzymes, a delivery vehicle (hyaluronidase), and stability excipients. The efforts in this project will set the groundwork to translate our subcutaneous approach into a clinically relevant product to complement or replace current intravenous treatments. This proposed study will focus on (Aim 1) developing highly concentrated subcutaneous GLA formulations and determining their stability, and (Aim 2) assessing the pharmacokinetic and biodistribution properties of subcutaneous GLA formulations against a standard intravenous dose in a rat model.